PREPARATION AND CHARACTERIZATION OF RHODAMINE-BASED IMMUNOCONJUGATES

BACKGROUND: Monoclonal antibody (MCAT)-based immunoconjugates have high potential in cancer immunotherapy, but at the moment the problem of developing effective methods for their preparation remains unresolved, especially in the case when a low-molecular-weight agent does not have chemical groups to attach to MCAT. This study is aimed at the synthesis of immunoconjugates based on a model substance, Rhodamine B (Rod):, which does not have active chemical groups for interaction with MCAT chemical groups. For this purpose, two Rod derivatives were synthesized: rhodamine-piperazine (Rod-piperazine) and its complex with the bifunctional agent SMCC (Rod-SMCC) and the subsequent synthesis of immunoconjugates based on them.

AIM: was to analyze the functional properties of immunoconjugates obtained on the basis of a modified rhodamine B molecule and a number of MCATs.

METHODS: Conjugation of MCAT with Rod-SMCC was performed using reduced disulfide groups of immunoglobulin G (IgG). Rod-piperazine was attached to oxidized IgG oligosaccharides. The synthesis was evaluated by spectrophotometric methods. The activity of the obtained conjugates was carried out using ELISA. The molecular heterogeneity of the conjugates obtained was analyzed by high-efficiency chromatography. Results. Using Rod-SMCC, it was possible to obtain immunoconjugates for thiol groups with 4 studied antibodies with maximum saturation for each antibody. The maximum inclusion, about 11.5 moles of Rod per mole of IgG, was obtained for MCAT ICO 204, about 5.5 for Trastuzumab. The resulting ICO 204 immunoconjugates retained antigen-binding activity comparable to native antibodies. The use of Rod-piperazine made it possible to obtain immunoconjugates with MCAT ICO 204 and Trastuzumab for oxidized carbohydrate residues of antibodies. The maximum density of Rhodamine B inclusion in these compounds did not exceed 3 moles per mole of IgG, which is probably due to significant steric limitations of the reaction.

CONCLUSION: As part of the work, the chemical synthesis of two derivatives of Rod, which initially did not have chemical groups for interaction with IgG, was carried out. Based on the obtained compounds, model immunoconjugates with a high degree of Rod inclusion were synthesized, which retain their antigen-binding activity. Thus, the possibility of using pharmacological substances with carboxyl groups in their structure that are not suitable for such synthesis to create immunoconjugates is shown, and the prospects of modifying pharmacological substances by the carboxyl group with piperazine and SMCC to obtain immunoconjugates are shown.